1. Field of the Invention
The present invention relates to a method for orientation-directed construction of a construct comprising at least two nucleic acid segments of interest. More particularly, the present invention provides a rapid and versatile method for efficient cloning of constructs by PCR enrichment of the desired orientation.
2. Prior Art
Genetic vectors are one of the most important basic tools for research throughout the world in the fields of medicine, biology and biotechnology. Almost all studies in these fields require use of genetic vectors. Such genetic vectors are not only needed in research; for example, in medicine, genetic vectors are a means for undertaking gene therapy of diseases with a genetic background. This promising field is at the forefront of the latest medical developments, when the sequences of the human genome have been finally determined, availing a fantastic amount of information on new genes usable for genetic repair to every researcher in the world. In addition, biotechnologists use genetic vectors for the introduction of genes into various organisms in order to produce large quantities of the protein expressed by these genes. Agriculture also make use of genetic vectors, mainly in producing transgenic plants of improved yield or improved resistance to both pets and pesticides and for the extension of shelf life of agricultural products.
Genetic vectors are shuttles containing a DNA sequence encoding genetic information, expressed in-vivo in organisms such as bacteria, yeast, cell aggregates and transgenic animals, or in in-vitro systems. Genetic vectors are in most cases circular DNA segments known as plasmids, which contain genetic information, but there are also other genetic vectors such as viruses and transpoisons tailored for the purpose. The information borne by the vectors is tailored to their biological objective and to the biological environment into which the vector is introduced. A genetic vector may contain genetic segments from different sources. For example, a sequence of a gene encoding a mamalian protein may be inserted into bacterial sequences in order to form a genetic vector expressing the human protein in bacteria, so that by culturing the bacteria one would obtain large quantities of that human protein.
Vector construction encompasses a variety of recombinant DNA technology methods based on digesting and re-ligating DNA fragments. Insertion of new genes into target DNA fragments, is one of the most widely known uses for recombinant DNA technology. This procedure includes digestion of the target fragment (which comprises the vector sequence) with a restriction enzyme. Similarly, the insert DNA, carrying the gene of interest, is digested with the same enzyme or with an appropriate enzyme that leaves the same 3′ or 5′ overhanging. In one type of restriction enzyme digestion, cleavage of both the target DNA and insert DNA leaves overlapping 3′ or 5′ nucleotide fragments on each end. These overlapping fragments or “sticky ends” are well known properties of some restriction enzymes.
Further methods of directly cloning DNA fragments into target DNA sequences are available, as the method described by Mead et al. [Bio/Technology 9:657 (1991)]. This method is based on the ability of Taq polymerase to inherently add deoxyadenosine (dATP) to the 3′ end of some newly synthesized duplex molecules described by Clark, J. M. [Nucleic Acids Research 20:9677 (1988)]. These single adenosine overhangs base pair with 3′ thymidine (dTTP) overhangs at the insertion site of a specially designed vector. It has been found that even single base pairs are sufficient for hydrogen bonding two nucleotide sequences together.
In yet another method of inserting a nucleotide fragment into a target DNA, known as blunt-end ligation, fragments to be ligated are digested using restriction enzymes which do not leave any 3′ or 5′ overhanging nucleotides at the enzyme splice site. These enzymes are known as “blunt-end” enzymes due to this feature of their enzymatic activity. After digestion, blunt-end restriction enzymes maintain single 5′ “terminal” phosphates on both sides of the restriction site. These terminal 5′ phosphates are required by DNA ligase for any subsequent religation of the digested DNA sequence.
None of the aforementioned methods permits a researcher to choose and enhance a specific orientation for the segment of insert. This presents a distinct disadvantage in that the insert DNA can position itself at either of two 5′ or 3′ orientations with respect to the target nucleotide sequence.
In most procedures the insert orientation is critical, for example, ligation of a promoter sequence to heterologous gene for obtaining subsequent gene expression or producing chimeric genes or proteins, requires ligation in the correct orientation.
Moreover, most of the vectors will self-ligate, without the insert, in case no direction is provided for the insert. Furthermore, up to 50% of the genes on average, when inserted into different vectors, will ligate in the wrong orientation. This dramatically reduces the overall experimental efficiency. For this reason, many methods have been devised for preferentially cloning insert DNA fragments into target sequences in a preferred orientation. These methods are commonly known as directional cloning techniques.
Directional cloning is usually performed initially by digesting the target nucleotide sequences with two different restriction enzymes, resulting in molecules having dissimilar DNA ends at the target insertion site. The insert DNA is also digested using the same two restriction enzymes, thereby producing two dissimilar DNA ends that corresponded to a specific orientation in the target insertion site. By following this procedure, the insert DNA could only bind the target sequence in the desired orientation.
Although this method has been widely used in the art, it does have disadvantages. For instance, digesting both the target DNA and insert DNA with multiple restriction enzymes is very time consuming. In addition, multiple enzyme digestions increase the risk that either the target or insert DNA sequence will be cleaved at an internal restriction site in a specific manner, or unspecifically (star activity) or alternatively, would not be cleaved at all. Problems associated with digesting the ends of DNA strands, also charachterize digestion based methods, such as that required in cutting the insert sequence. Furtheremore, digestion using two different restriction endonucleases, requires changing buffers between each reaction, and decrease the probability of proper digestion of the DNA sequence. Moreover, the use of restriction enzymes in vector designing is a limiting factor in inserting sequences that may affect the flexibility of the desired construct.
Many investigators have attempted to improve methods relating to directionally cloning of DNA fragments, as detailed above. One of the most widely used procedures involving directional ligation relates to subcloning DNA fragments that have been amplified by the polymerase chain reaction (PCR).
The most common method for cloning PCR products involves incorporation of flanking restriction sites onto the ends of primer molecules. The PCR cycling is carried out and the amplified DNA is then purified, restricted with an appropriate endonuclease(s) and ligated to a compatible vector preparation. Thus, typical PCR cloning methods require preparation of PCR primer molecules attached to “add on” base sequences having a preferred restriction recognition sequence. Also, these methods can result in unintended internal restriction of uncharacterized or polymorphic sequences. Such limitations of previous methods add to the cost and complexity of cloning PCR products routinely.
Moreover, this protocol has the same drawbacks as the aforementioned double digestion method. In addition, the PCR primers have more bases to accommodate the restriction site that results in added expense for PCR primers. Additionally, this method is limited by the often inefficient cleavage of restriction endonuclease cleavage sites near an end of a double-stranded nucleic acid.
Another method of directionally cloning an insert into a target sequence uses Exonuclease III [Kaluz, et al., Nucleic Acids Research, 20(16):4369-4370 (1992)] to create the “sticky ends”. In the method described by Kaluz et al., insert DNA fragments were digested with Exonuclease III. The number of nucleotides that Exonuclease III digests from the 3′ end of DNA per minute is well known. After a timed digestion, the insert fragments were left with 5′ overlapping nucleotide tails. These tails were engineered so that the 5′ ends would only hybridize in one orientation upon base pairing to the target plasmid DNA molecule.
Nevertheless, the Exonuclease III method is specifically time-dependent and the enzyme continues to digest DNA as long as the reaction is incubated. For this reason, Exonuclease III might potentially digest through the end nucleotides and into the coding region of the insert DNA sequence, prompting an undesiered experimental result.
In yet another method, production of sticky ends is performed using the 3′ exonuclease activity of T4 DNA polymerase [Kuijper, J. L., et al. Gene 112: 147-155 (1992)], a further method of directionally cloning PCR generated fragments into a target DNA sequence based on incorporation of uracil into the PCR primers, followed by treatment with uracil-N-glycosylase [Nisson, P. C., et al. PCR Methods and Applications 1:120-123 (1991)].
Another example is disclosed in U.S. Pat. No. 5,523,221, directed at a method for directionally cloning an insert DNA sequence into a target DNA sequence. This method includes generating a monophosphorylated target DNA sequence and a monophosphorylated insert DNA sequence, followed by combining the insert DNA sequence, preferably with DNA ligase, with the target sequence. Thus, the insert sequence can only ligate in one orientation with respect to the target sequence. Preferably, the target DNA sequence is a plasmid. This method uses the calf intestinal alkaline phosphatese (CIAP) to produce monophosphate target DNA. The method has several disadvantage, the use of monophosphorylated target DNA and insert DNA significantly reduces the ligation efficiency and requires multiple restriction enzymes digestion.
All of the above directional cloning methods require multiple restriction enzyme digestion, addition of extra nucleotides to the insert, or are expensive and time consuming. For these reasons, there is a need for a simple, efficient, rappid and inexpensive method of directionally cloning a DNA fragment into its target sequence.
To solve the above mentioned complexity of endonucleases involvement, PCR technology has been used to engineer hybrid (chimeric) genes without the need to use restriction enzymes in order to segment the gene prior to hybrid formation. In this approach, fragments of the different genes that are intended to form the hybrid are generated in separate polymerase chain reactions. The primers used in these separate reactions are designed so that the ends of the different products of the separate reactions contain complementary sequences. When these separately produced PCR products are mixed, denatured and reannealed, the strands having matching sequences at their 3′-ends overlap, and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are spliced together to form the hybrid gene. Thus, this method requires specific primers each time one wishes to construct a chimeric molecule that contains the same fragments in different location or orientation. Furthermore, it does not allow a straightforward means to generate inverted or directly repeated DNA sequences [Horton, et al., Gene 77:61-68 (1989)].
Overall, current methods used for the construction of genetic vectors involve multi-step processes which are very time consuming. On average, the time required to introduce one genetic insert into an existing plasmid is more than three days. When a genetic vector is required to be assembled of several different genetic segments, its construction may take several weeks and more. In addition, when a genetic segment must be incorporated in a sequence in a definite orientation, the process complexity is increased and the products require a longer scanning time.
Ligase-free subcloning was reported as a rapid subcloning method [Shuldiner A., et al., Nucleic Acid Research, 18:1920 (1990) and Analytical Biochemistry 194:9-15 (1991)]. This procedure is performed by incorporating into the PCR primers sequences at the 5′ ends that result in a PCR product whose 3′ ends are complementary to the 3′ ends of the recipient linearized plasmid. The PCR product and the plasmid are spliced together in a second PCR reaction in which Taq polymerase extends the complementary overlapping 3′ ends. However, this method as well has several disadvantage compares to the method of the present invention. The primers designed for the ligase-free method should comprise sequences complementary to both segments of interest, and therefore these primers are quite long (about 50 nucleotides) expensive and having increased potential of non-specific annealing to the atmplate, whereas the primers designed for the present method should have the minimal length permitting specific annealing to the template. Furtheremore, the segments as well as the primers prepared for the present invention can be used for construction of different constructs. For example, a segment comprising an origin of replication and selectable marker, or even a certain reporter gene, can be ligated to different segments of interest in different constructs. These combined products can be enriched for the desired orientation by using the same primers, whereas the ligase-free method of Shuldiner et al., is limited in the specific direction and combination of segments. Thus, any different construct will require production of different products and synthesis of different primers, whereas the present invention is much more versatile and economic. Other disadvantages of the Shuldiner et al., ligase-free method are the necessity of extensive optimization of the number of PCR cycles of the second-stage PCR, the temperature at which hetrologous reannealing and cyclization, the concentration of the NaCl. Most importantly, the reported efficiency of Shulinder et al., method is about 50% of the total number of the colonies, whereas the efficiency of the method of the present invention is about 95%. Furtheremore, successful subcloning with the Shulinder et al., method has been accomplished for PCR products as large as 1.7 kb. And finally, two of the primers used by the method of Shulinder et al., are complementary at their 3′ end to the target sequence and at their 5′ end to the plasmid. Thus, in order to insert the same fragment into two different plasmids about eight primers are required, whereas only six primers are needed for the OER method of the invention.
The present invention is based on the development of a rapid method designed to shorten and facilitate the process of constructing genetic vectors and streamlines this process, by providing genetic vectors aligned with their sequences in the proper orientation, without need for extensive scanning. The central process carried out in the method of the invention is designated as Orientation Enrichment Reaction (OER). In this process there is enrichment at every cycle, enreaches towards those vectors containing genetic segments aligned in the proper direction, so that at the end of the process a genetic vector, in which all segments are properly aligned, is obtained. Each cycle is made up of a phase of polymerase chain reaction, followed by product cleaning and then a stage of segment ligation. According to the method of the invention, an unlimited number of DNA segments may be ligated together whereby the size of the final segment obtained is the only restriction.
The method of the invention significantly shortens the time needed to construct a genetic vector, rendering construction of the vectors very easy and more versatile. On average, eight genetic segments may be ligated together overnight. When the directionality of the genetic vectors is examined, about 95% of them turn out to contain the required sequence with the proper orientation.